A chromosome-scale genome provides new insights into the typical carotenoid biosynthesis in the important red yeast Rhodotorula glutinis QYH-2023 with anti-inflammatory effects.

Rhodotorula spp. has been studied as one powerful source for a novel cell factory with fast growth and its high added-value biomolecules. However, its inadequate genome and genomic annotation have hindered its widespread use in cosmetics and food industries. Rhodotorula glutinis QYH-2023, was isolated from rice rhizosphere soil, and the highest quality of the genome of the strain was obtained at chromosome level (18 chromosomes) than ever before in red yeast in this study. Comparative genomics analysis revealed that there are more key gene copies of carotenoids biosynthesis in R. glutinis QYH-2023 than other species of Rhodotorula spp. Integrated transcriptome and metabolome analysis revealed that lipids and carotenoids biosynthesis was significantly enriched during fermentation. Subsequent investigation revealed that the over-expression of the strain three genes related to carotenoids biosynthesis in Komagataella phaffii significantly promoted the carotenoid production. Furthermore, in vitro tests initially confirmed that the longer the fermentation period, the synthesized metabolites controlled by R. glutinis QYH-2023 genome had the stronger anti-inflammatory properties. All of the findings revealed a high-quality reference genome which highlight the potential of R. glutinis strains to be employed as chassis cells for biosynthesizing carotenoids and other active chemicals.

Integrated Transcriptomics and Metabolomics Analysis Reveal the Regulatory Mechanisms Underlying Sodium Butyrate-Induced Carotenoid Biosynthesis in Rhodotorula glutinis.

Sodium butyrate (SB) is a histone deacetylase inhibitor that can induce changes in gene expression and secondary metabolite titers by inhibiting histone deacetylation. Our preliminary analysis also indicated that SB significantly enhanced the biosynthesis of carotenoids in the Rhodotorula glutinis strain YM25079, although the underlying regulatory mechanisms remained unclear. Based on an integrated analysis of transcriptomics and metabolomics, this study revealed changes in cell membrane stability, DNA and protein methylation levels, amino acid metabolism, and oxidative stress in the strain YM25079 under SB exposure. Among them, the upregulation of oxidative stress may be a contributing factor for the increase in carotenoid biosynthesis, subsequently enhancing the strain resistance to oxidative stress and maintaining the membrane fluidity and function for normal cell growth. To summarize, our results showed that SB promoted carotenoid synthesis in the Rhodotorula glutinis strain YM25079 and increased the levels of the key metabolites and regulators involved in the stress response of yeast cells. Additionally, epigenetic modifiers were applied to produce fungal carotenoid, providing a novel and promising strategy for the biosynthesis of yeast-based carotenoids.

Agricultural wastes: a new promising source for phenylalanine ammonia-lyase as anticancer agent.

The present study aims to investigate the physicochemical characteristics of phenylalanine ammonia-lyase (PAL) extracted from agricultural waste and its potential use as an anticancer agent in comparison to microbial PAL. We extracted and partially purified PAL from agricultural waste sources. We assessed the temperature and pH range of PAL and determined enzyme kinetics parameters including Michaelis constants (Km), maximum velocity (Vmax), and specificity constant values (Vmax/Km). Additionally, we examined the effects of different storage temperatures on PAL activity. In our analysis, we compared the efficacy of agricultural waste-derived PAL with PAL from Rhodotorula glutinis. The results demonstrated that PAL extracted from agricultural waste exhibited significantly higher specific activity (Vmax/Km) compared to its microbial counterpart. The agricultural waste-derived PAL displayed a stronger affinity for phenylalanine, as indicated by a lower Km value than the microbial PAL did. Furthermore, PAL from agricultural waste maintained activity across a broader temperature and pH range (15-75 °C, pH 5-11), in contrast to microbial PAL (20-60 °C, pH 5.5-10). Importantly, the PAL derived from agricultural waste exhibited superior stability, retaining over 90% of its activity after 6 months of storage at room temperature (25 °C), whereas microbial PAL lost more than 70% of its activity under similar storage conditions. In anticancer experiments against various cancer cell lines, agricultural waste-derived PAL demonstrated greater anticancer activity compared to microbial PAL. These findings suggest that PAL sourced from agricultural waste has the potential to be a safe and effective natural anticancer agent.

Fermentative Production of ß-Carotene from Sugarcane Bagasse Hydrolysate by Rhodotorula glutinis CCT-2186.

Β-Carotene is a red-orange pigment that serves as a precursor to important pharmaceutical molecules like vitamin A and retinol, making it highly significant in the industrial sector. Consequently, there is an ongoing quest for more sustainable production methods. In this study, glucose and xylose, two primary sugars derived from sugarcane bagasse (SCB), were utilized as substrates for ß-carotene production by Rhodotorula glutinis CCT-2186. To achieve this, SCB underwent pretreatment using NaOH, involved different concentrations of total solids (TS) (10%, 15%, and 20%) to remove lignin. Each sample was enzymatically hydrolyzed using two substrate loadings (5% and 10%). The pretreated SCB with 10%, 15%, and 20% TS exhibited glucose hydrolysis yields (%wt) of 93.10%, 91.88%, and 90.77%, respectively. The resulting hydrolysate was employed for ß-carotene production under batch fermentation. After 72 h of fermentation, the SCB hydrolysate yielded a ß-carotene concentration of 118.56 ± 3.01 mg/L. These findings showcase the robustness of R. glutinis as a biocatalyst for converting SCB into ß-carotene.

Utilization of olive mill wastewater for selective production of lipids and carotenoids by Rhodotorula glutinis.

Olive mill wastewater (OMW) is a zero-cost substrate for numerous value-added compounds. Although several studies on the production of lipids and carotenoids by Rhodotorula glutinis in OMW exist, none of them has specifically focused on the conditions for a target lipid or carotenoid. This study presents cultivation conditions that selectively stimulate the cell biomass, individual carotenoids and lipids. It was found that supplemental carbon and nitrogen sources as well as illumination affected cell biomass the most. High temperature, low initial pH, illumination, lack of urea and presence of glycerol stimulated the lipid synthesis. The highest total lipid content obtained in undiluted OMW supplemented with urea was 11.08 ± 0.17% (w/w) whilst it was 41.40 ± 0.21% (w/w) when supplemented with glycerol. Moreover, the main fatty acid produced by R. glutinis in all media was oleic acid, whose fraction reached 63.94 ± 0.58%. Total carotenoid yield was significantly increased with low initial pH, high temperature, illumination, certain amounts of urea, glycerol and cultivation time. Up to 192.09 ± 0.16 µg/g cell carotenoid yield was achieved. Torularhodin could be selectively produced at high pH, low temperature and with urea and glycerol supplementation. To selectively induce torulene synthesis, cultivation conditions should have low pH, high temperature and illumination. In addition, low pH, high temperature and urea supplementation served high production of ß-carotene. Up to 85.40 ± 0.76, 80.67 ± 1.40 and 39.45 ± 0.69% of torulene, torularhodin and ß-carotene, respectively, were obtained under selected conditions. KEY POINTS: • Cultivation conditions selectively induced target carotenoids and lipids • 41.40 ± 0.21% (w/w) lipid content and 192.09 ± 0.16 µg/g cell carotenoid yield were achieved • Markedly high selectivity values for torularhodin and torulene were achieved.

Valorization of food wastes with a sequential two-step process for microbial ß-carotene production: A zero waste approach.

Here, two consecutive ß-carotene fermentation processes were carried out with Rhodotorula glutinis yeast in the growth media obtained from orange and grape wastes. Firstly, waste biomasses were subjected to hot water extraction. Effects of waste type, drying pretreatment, particle size and solid/liquid ratio on the total concentration and yield of sugars recovered were tested. The highest sugar concentration was obtained by the hot water extraction of fresh grape pomace as 61.2 g total reducing sugars (TRS)/L at a solid/liquid ratio of 100 g/L. In the first fermentation process, effect of solid/liquid ratio (initial TRS concentration) on ß-carotene production pattern of R. glutinis was investigated in the media obtained directly by hot water extraction of the wastes. Microorganism and ß-carotene concentrations increased with increasing solid/liquid ratio (range 10-100 g/L), and the microbial growth data fit the Monod model well for all cases. Maximum ß-carotene concentration in the growth medium obtained from hot water extraction of 100 g/L of grape pomace was determined as 5988.6 mg/L. In the second fermentation process, ß-carotene was produced in the acid hydrolysate of extraction residues. 10.1 g/L and 6.7 g/L of TRS was obtained after acid hydrolysis of orange and grape residues, respectively, and the highest ß-carotene concentration of 370.0 mg/L was found in the medium of hydrolyzed orange peel extraction residue. Total ß-carotene production increased to 1777.1 and 3279.6 mg/L (26% and 4.9% of increase) after the second fermentation step. 85.3% and 80.2% of reduction in orange and grape waste weights were observed at the end of the process, which was an indicator of efficient waste biomass disposal. Two sequential ß-carotene fermentation steps offered significant advantages in terms of both efficiency and a zero waste approach.

Antimicrobial and anti-biofilm effects of carotenoid pigment extracted from Rhodotorula glutinis strain on food-borne bacteria.

Background and Objectives: Carotenoid pigments are among the most important pigments and have many applications in various food, cosmetics, hygiene industries and biotechnology. These pigments are produced by plants and microorganisms including Rhodotorula spp. This research intended to study the antimicrobial and antibiofilm effects of the carotenoid pigment from Rhodotorula glutinis on food spoilage bacteria (Staphylococcus aureus and Salmonella Typhimurium). Materials and Methods: The R. glutinis was isolated from milk samples of cows with mastitis and ITS sequence-based typing was performed on them. After extracting the pigment from R. glutinis, its purity was examined using thin-layer chromatography. Following that, the broth microdilution method was used to evaluate antimicrobial effects of the pigment and MtP assay and subsequently scanning electron microscopy were used to assess the antibiofilm effects. In addition, the sub-MIC effects of the pigment on expression of quorum-sensing (QS) genes in S. Typhimurium isolates (sdiA and luxS) and S. aureus isolates (hld) were studied. Finally, the degree of toxicity of the pigment was analyzed using the MTT assay. Results: ITS sequence analysis of R. glutinis revealed that the recently separated isolates exhibited strong differences with the strains recorded in NCBI database in genetic structure. The pigment produced by R. glutinis had strong antimicrobial effects and its mean MIC against S. Typhimurium isolates (17.0 µl.ml-1) was higher than the mean MIC against the S. aureus isolates (4.1 µl.ml-1). Electron microscope images and real-time observations indicated that the sub-MIC values of the pigment suppressed biofilm formation by suppressing expression of QS genes. In addition, the mentioned pigment at high MIC concentrations did not have toxic effects on Vero cells. Conclusion: This research suggests that R. glutinis pigment is effective in destroying the planktonic form of food spoilage bacteria and degrading food spoilage biofilm-forming bacteria. Moreover, considering the low toxicity level of R. glutinis pigment for eukaryotic cells, we can suggest its use as a natural antibacterial preservative in various food materials.

Study on the fermentation effect of Rhodotorula glutinis utilizing tofu whey wastewater and the influence of Rhodotorula glutinis on laying hens.

Background: Tofu whey wastewater (TWW) is the wastewater of tofu processing, which is rich in a variety of nutrients. Rhodotorula glutinis can make full use of TWW to ferment and reproduce yeast cells, produce carotenoids and other nutrients, improve the utilization value of TWW, and reduce environmental pollution and resource waste. Methods: In this study, the nutrient composition changes of TWW treated by Rhodotorula glutinis were analyzed to reformulate TWW medium, and the optimal composition and proportion of TWW medium that can improve the biomass and carotenoids production of Rhodotorula glutinis were explored. Meanwhile, the Rhodotorula glutinis liquid obtained under these conditions was used to prepare biological feed for laying hens, and the effect of Rhodotorula glutinis growing on TWW as substrate on laying performance and egg quality of laying hens were verified. Results: The results showed that the zinc content of TWW after Rhodotorula glutinis fermentation increased by 62.30%, the phosphorus content decreased by 42.31%, and the contents of vitamin B1, B2 and B6 increased to varying degrees. The optimal fermentation conditions of Rhodotorula glutinis in the TWW medium were as follow: the initial pH was 6.40, the amount of soybean oil, glucose and zinc ions was 0.80 ml/L, 16.32 g/L, and 20.52 mg/L, respectively. Under this condition, the biomass of Rhodotorula glutinis reached 2.23 g/L, the carotenoids production was 832.86 µg/g, and the number of effective viable yeast count was 7.08 × 107 cfu/ml. In addition, the laying performance and egg quality of laying hens fed Rhodotorula glutinis biological feed were improved. Discussion: In this study, we analyzed the composition changes of TWW, optimized the fermentation conditions of Rhodotorula glutinis in TWW medium, explored the influence of Rhodotorula glutinis utilizing TWW on laying layers, and provided a new idea for the efficient utilization of TWW.

Novel flavonoid C-8 hydroxylase from Rhodotorula glutinis: identification, characterization and substrate scope.

BACKGROUND: The regioselective hydroxylation of phenolic compounds, especially flavonoids, is still a bottleneck of classical organic chemistry that could be solved using enzymes with high activity and specificity. Yeast Rhodotorula glutinis KCh735 in known to catalyze the C-8 hydroxylation of flavones and flavanones. The enzyme F8H (flavonoid C8-hydroxylase) is involved in the reaction, but the specific gene has not yet been identified. In this work, we present identification, heterologous expression and characterization of the first F8H ortho-hydroxylase from yeast. RESULTS: Differential transcriptome analysis and homology to bacterial monooxygenases, including also a FAD-dependent motif and a GD motif characteristic for flavin-dependent monooxygenases, provided a set of coding sequences among which RgF8H was identified. Phylogenetic analysis suggests that RgF8H is a member of the flavin monooxygenase group active on flavonoid substrates. Analysis of recombinant protein showed that the enzyme catalyzes the C8-hydroxylation of naringenin, hesperetin, eriodyctiol, pinocembrin, apigenin, luteolin, chrysin, diosmetin and 7,4'-dihydroxyflavone. The presence of the C7-OH group is necessary for enzymatic activity indicating ortho-hydroxylation mechanism. The enzyme requires the NADPH coenzyme for regeneration prosthetic group, displays very low hydroxyperoxyflavin decupling rate, and addition of FAD significantly increases its activity. CONCLUSIONS: This study presents identification of the first yeast hydroxylase responsible for regioselective C8-hydroxylation of flavonoids (F8H). The enzyme was biochemically characterized and applied in in vitro cascade with Bacillus megaterium glucose dehydrogenase reactions. High in vivo activity in Escherichia coli enable further synthetic biology application towards production of rare highly antioxidant compounds.

Transcriptomic and Metabolomic Analyses Provide Insights into the Enhancement of Torulene and Torularhodin Production in Rhodotorula glutinis ZHK under Moderate Salt Conditions.

Carotenoids are a group of tetraterpene pigments widely used in the food, pharmaceutical, and cosmetic industries. Torulene, torularhodin, and ß-carotene, three principal carotenoids synthesized by Rhodotorula glutinis ZHK, possess strong health-promoting properties such as antioxidant, provitamin A, and antitumor. Here, the effect of different salt conditions on carotenoids production of R. glutinisZHK was investigated. The results showed that the total carotenoids were significantly enhanced in 0.5 M (3.91 mg/L) and 0.75 M (5.41 mg/L) NaCl treatments than that in 1.0 M (0.35 mg/L) and control (1.42 mg/L) after 120 h of cultivation. Of which, the increase in torulene and torularhodin production acts as the main contributor to the enhancement of total carotenoids. Transcriptome profiling revealed that salt stress efficiently promotes the gene expression of crtI, which could explain the molecular mechanisms of the enhanced torulene and torularhodin production under salt stress. Further experiments indicated that torulene and torularhodin play an important role in quenching excrescent reactive oxygen species induced by salt stress. Together, the present study reports an effective strategy for simultaneously improving torulene and torularhodin production in R. glutinis ZHK.

Pike-Perch (Sander lucioperca) and Rainbow Trout (Oncorhynchus mykiss) Fed with an Alternative Microorganism Mix for Reducing Fish Meal and Oil-Fishes' Growth Performances and Quality Traits.

In the last decades, several plant-based materials were used for the substitution of fish meal and oil in aquaculture. The present study evaluated the fish quality and the sensory differences of rainbow trout (Oncorhynchus mykiss) and pike-perch (Sander lucioperca) from three different feeding groups, which were fed a commercially available industrial (standard) diet, a control diet, and a special microorganism-based feed mix. This feed mainly consisted of a mix made of Rhodotorula glutinis, Crypthecodinium cohnii, and Arthrospira sp. and had 50% less fish meal and fish oil compared to typical control diets. At the beginning, the pike-perch population was six months old, and the rainbow trout population was 15 months old. The feeding study duration was 16 weeks and every four weeks the growth performance and several morphometric parameters were recorded. Afterwards, sensory evaluation took place to identify possible trends. Sensory evaluation revealed that the rainbow trout groups did not show any significant differences to the standard and control fish fillets with regard to odor, texture, and taste. The effects on rainbow trout growth performances and carcass parameters were similar to the standard group. The feed mix was not optimal for pike-perch farming, which was also reflected by significantly adversely affected growth performance and carcass parameters. The sensorial evaluation showed an opposite trend: here, only small differences in the fillets from the feed mix and standard/control diet were observed.

Harvesting of Rhodotorula glutinis via Polyaluminum Chloride or Cationic Polyacrylamide Using the Extended DLVO Theory.

Polyaluminum chloride (PAC) and cationic polyacrylamide (CPAM) play a crucial role for separating microorganisms from bulk media. However, the mechanism of adsorption between cells and flocculants remain to be further defined to improve the flocculation efficiency (FE) in extreme conditions. This study conducted the flocculation process of Rhodotorula glutinis induced by PAC and CPAM, firstly. The result demonstrated that CPAM possessed more efficient harvesting ability for R. glutinis compared to PAC. The difference of flocculation capacity was then thermodynamically explained by the extended DLVO (eDLVO) theory; it turned out that the poor harvesting efficiency of PAC was attributed to lacking of binding sites as well as low adsorption force within particles. Based on this, the FE of PAC to R. glutinis was mechanically enhanced to 99.84% from 32.89% with 0.2 g/L CPAM modification at an optimum pH of 9. Also, the paper will play a guiding role in the treatment of inorganic salt ions and organic matters in wastewater.

Farnesol and tyrosol: novel inducers for microbial production of carotenoids and prodigiosin.

This study was performed to elucidate the effects of two fungal quorum sensing molecules (tyrosol and farnesol) on carotenoid synthesis in the yeast Rhodotorula glutinis and prodigioin synthesis in the bacterium Serratia marcencens. Farnesol or tyrosol was directly added to the flask cultures at the beginning (immediately after inoculation with the preculture) of day 1 or the beginning (49th h) of day 3. The results demonstrated that tyrosol supplementation increased the synthesis of carotenoids but farnesol supplementation increased the synthesis of prodigiosin. It was found that adding farnesol or tyrosol into the culture on day 3 compared to day 1 caused more increments in pigment synthesis. The maximum increase (fivefold) in the synthesis of prodigiosin was achieved with 200 µL/L farnesol supplementation, whereas the maximum increase (2.13 fold) in the synthesis of carotenoids was achieved with 4 mg/L tyrosol supplementation. This is the first report about the effects of fungal quorum sensing molecules (farnesol and tyrosol) on the synthesis of carotenoids and prodigiosin in microorganisms. Due to non-human toxicity and low price and of farnesol and tyrosol, these molecules can be used as novel inducers for large-scale production of microbial pigments.

Effect of mixed culture of yeast and microalgae on acetyl-CoA carboxylase and Glycerol-3-phosphate acyltransferase expression.

In recent years, some studies have reported that co-culturing green algae and yeast improve lipid and biomass concentration. In this study, a co-culture of the oleaginous yeast Rhodotorula glutinis and the microalgae Chlorella vulgaris was consequently conducted with inoculation of microalga and yeast in growth and stationary phases, respectively. For the first time, the expression of two pivotal enzymes in fatty acids synthetic pathway, acetyl-CoA carboxylase and Glycerol-3-phosphate acyltransferase, was evaluated. To evaluate the synergistic impacts of the mixed culture on the enzymes expression, several co-culture models were designed, including the use of different ratio of microalgae to yeast or the use of residual cell-free medium of yeast; a positive impact on enzymes overexpression was shown in the case of the co-culture of the two microorganisms, and when the remaining cell-free medium of yeast was added to the microalgal culture. The results of in vitro co-culture demonstrated increased 6- and 5-fold of nervonic acid (C24:1) and behenic acid (C22:0) concentrations, respectively, in 2:1 microalgae to yeast co-culture as compared to the monoculture batches. Addition of yeast residual cell-free medium in the 2:1 ratio to the microalgal culture enhanced 9 and 6 times nervonic acid (C24:1) and behenic acid (C22:0) amounts, respectively.

The characteristics of gelation of myofibrillar proteins combined with salt soluble Rhodotorula glutinis proteins by enzymatic crosslinking.

Some microbial single-cell proteins are capable of producing synergistic crosslinking interactions with edible proteins by rational regulation. Herein, we reported that salt soluble proteins (RGP) extracted from Rhodotorula glutinis in an alkaline and saline system may combine with myofibrillar proteins (MP) by transglutaminase (TG) polymerization to form improvable irreversible thermal co-gels. The combination of MP, RGP, and TG, namely restructured MP gels, led to significantly enhanced water holding capacity (WHC), up to 90.76 ± 1.88% (% of retained water) and textural properties (hardness, springiness, and adhesiveness) as well as decreases of 'gauche-gauche-gauche' SS bonds and α-helix conformations and increases of 'gauche-gauche-trans' SS bonds and ß-fold conformations, compared to MP and MP-RGP groups. Differential scanning calorimetry analysis validated that thermostability of myosins and actins from MP was reduced after using RGP, TG, and their combination, and unfolding and denaturation of myosin occurred easily during thermal co-gelation by TG and/or RGP.

Genomics and lipidomics analysis of the biotechnologically important oleaginous red yeast Rhodotorula glutinis ZHK provides new insights into its lipid and carotenoid metabolism.

BACKGROUND: Rhodotorula glutinis is recognized as a biotechnologically important oleaginous red yeast, which synthesizes numerous meritorious compounds with wide industrial usages. One of the most notable properties of R. glutinis is the formation of intracellular lipid droplets full of carotenoids. However, the basic genomic features that underlie the biosynthesis of these valuable compounds in R. glutinis have not been fully documented. To reveal the biotechnological potential of R. glutinis, the genomics and lipidomics analysis was performed through the Next-Generation Sequencing and HPLC-MS-based metabolomics technologies. RESULTS: Here, we firstly assemble the genome of R. glutinis ZHK into 21.8 Mb, containing 30 scaffolds and 6774 predicted genes with a N50 length of 14, 66,672 bp and GC content of 67.8%. Genome completeness assessment (BUSCO alignment: 95.3%) indicated the genome assembly with a high-quality features. According to the functional annotation of the genome, we predicted several key genes involved in lipids and carotenoids metabolism as well as certain industrial enzymes biosynthesis. Comparative genomics results suggested that most of orthologous genes have underwent the strong purifying selection within the five Rhodotorula species, especially genes responsible for carotenoids biosynthesis. Furthermore, a total of 982 lipids were identified using the lipidomics approaches, mainly including triacylglycerols, diacylglyceryltrimethylhomo-ser and phosphatidylethanolamine. CONCLUSION: Using whole genome shotgun sequencing, we comprehensively analyzed the genome of R. glutinis and predicted several key genes involved in lipids and carotenoids metabolism. By performing comparative genomic analysis, we show that most of the ortholog genes have undergone strong purifying selection within the five Rhodotorula species. Furthermore, we identified 982 lipid species using lipidomic approaches. These results provided valuable resources to further advance biotechnological applications of R .glutinis.

Surfactant-assisted in situ transesterification of wet Rhodotorula glutinis biomass.

In situ transesterification of oleaginous microbes with short chain alcohol has been developed as a renewable process for the production of biodiesel. Dry biomass is often a requisite for the process to avoid the adverse effect of water on the productivity. As a consequence, large amount of energy consumption is required for prior biomass drying. In this study, the wet biomass of Rhodotorula glutinis, an oleaginous yeast, was used directly in in situ transesterification without biomass drying. The reaction conditions were optimized for the production of fatty acid methyl esters (FAME) and the effects of adding different surfactants were also studied. The highest FAME yield of 110% was achieved with a methanol loading of 1:100 at 90°C for 8 h as catalyzed by 0.36 M H2SO4, and the FAME content was 97%, which meets the 96.5% specified in both European biodiesel standards and Taiwanese biodiesel standards. The addition of 50 mM 3-(N,N-dimethylmyristylammonio)propanesulfonate (3-DMAPS, a zwitterionic surfactant) improved the FAME yield from 69% to 83%, which was obtained with a low methanol loading of 1:10 at 90°C for 10 h. Hence, the production of FAME with wet biomass under optimized reaction conditions was as effective as that with the dry form. This clearly indicates that using wet R. glutinis as the feedstock is feasible for the production of biodiesel by in situ transesterification.

Lipid Production by Rhodotorula glutinis in Continuous Cultivation with a Gravity Sedimentation System.

Lipid accumulation is generally believed to be a partially growth-coupled biochemical process that results in differences in lipid content between different cells. To separate lipid-rich cells and increase the cellular biomass in bioreactors during the cultivation of the oleaginous yeasts, a gravity sedimentation system (GSS) is coupled to a bioreactor. The dilution rate (D) and the ratio of the outflow rate from the outlet of the GSS to the inflow rate into the bioreactor (B) were evaluated. The biomass in the bioreactor with GSS increased by 16.3% and 30.6% at D values of 0.05 h-1 (B = 0.25) and 0.02 h-1 (B = 0.5), respectively. Interestingly, cells containing 39.3% lipids were obtained from the outlet of the GSS (D = 0.02 h-1, B = 0.5), and the lipid content increased by 7.8% compared to that of the bioreactor. The results indicated that use of a GSS is a very effective method for increasing the cell concentration and separation of lipid-rich cells.

Fatty acids profiles and estimation of the biodiesel quality parameters from Rhodotorula spp. from Antarctica.

OBJECTIVE: Oleaginous yeasts are a renewable and alternative source of oil for third-generation biodiesel. This work aimed to evaluate the effects of glucose concentration (30-100 g L-1) on growth, lipid synthesis, and fatty acids (FA) profile of three Rhodotorula spp. (R. glacialis R15, R. glutinis R4, and R. glutinis R48) isolated from Antarctica, and estimate the key quality parameters of the biodiesel produced by yeasts to confirm their potential as feedstocks for third-generation biodiesel synthesis. RESULTS: Yeasts accumulated 50-69.5% of lipids (w/w) under nitrogen-limitation and glucose-excess (C/N = 40-133). Glucose concentration increase influenced positively lipid accumulation (69.5% w/w) and FA profile of R. glacialis R15. Lipid accumulation (53% on average) of R. glutinis strains was not significantly affected by glucose concentration; content of saturated (~ 30%) and polyunsaturated FA (~ 29-30%) was slightly influenced. FA profiles of lipids synthesized by R15, R4, and R48 are similar to vegetable oils used in biodiesel industry with C16 and C18 FA (95-99%) as the major components, and contain mainly oleic (C18:1), palmitic (C16:0), and linoleic (C18:2) acids, which are suitable for biodiesel synthesis. Estimated fuel properties for biodiesel produced by R15, R4, and R48 satisfied all the criteria established by ASTM D6751 and EN 14214 with good cetane number, iodine value, and oxidation stability. An improvement in biodiesel quality of R15 was observed with the glucose increase. The best global properties of biodiesel from R4 were obtained with 30 g L-1 of glucose. CONCLUSIONS: Rhodotorula spp. from Antarctica are promising candidates for third-generation biodiesel synthesis.

Organic-solvent-free extraction of carotenoids from yeast Rhodotorula glutinis by application of ultrasound under pressure.

The extraction of Rhodotorula glutinis carotenoids by ultrasound under pressure (manosonication) in an aqueous medium has been demonstrated. The influence of treatment time, pressure, and ultrasound amplitude on R. glutinis inactivation and on the extraction of carotenoids was evaluated, and the obtained data were described mathematically. The extraction yields were lineal functions of those three parameters, whereas inactivation responded to a more complex equation. Under optimum treatment conditions, 82% of carotenoid content was recovered. Extraction of carotenoids in an aqueous medium was attributed to the capacity of ultrasound for cell disruption and emulsification. Cavitation caused the rupture of cell envelopes and the subsequent formation of small droplets of carotenoids surrounded by the phospholipids of the cytoplasmic membrane that would stabilize the emulsion. Analysis of the dispersed particle size of the extracts demonstrated that a fine, homogeneous emulsion was formed after treatment (average size: 230 nm; polydispersity <0.22). This research describes an innovative green process for extracting carotenoids from fresh biomass of R. glutinis in which only two unit operations are required: ultrasonic treatment, followed by a centrifugation step to discard cell debris. The extract obtained thanks to this procedure is rich in carotenoids (25 mg/L) and could be directly incorporated as a pigment in foods, beverages, and diet supplements; it can also be utilized as an ingredient in drugs or cosmetics.